Transporter proteins constitute a significant fraction of membrane-bound proteins, which account for approximately 30% of total proteins found in humans. About half of the membrane-bound transporters reported to date have been discovered within the past 5 years. The role of membrane transporters located in various organs and tissues in drug delivery and their disposition in vivo is just beginning to be appreciated. For example, a major challenge in clinical pharmacology is to elucidate the often observed, wide inter-individual variability in pharmacokinetics. Recently, Greiner et al. (J. Clin. Invest. 104:147 (1999)) demonstrated an inter-individual variation in the intestinal expression of the multidrug resistance transporter protein, MDR1 or P-glycoprotein, that resulted in a 7-fold range in the oral bioavailability of a P-glycoprotein (pgp) substrate, digoxin, in healthy human subjects. Therefore, it is important to understand the role of transporters in the disposition of currently available drugs, as well as to develop the capability of predicting the contribution of membrane transporters in the disposition of new molecular entities under development.
There are two well-characterized major ATP-binding cassette (ABC) superfamily members, ABC-B and ABC-C, involved in conferring drug resistance to cancer cells (Higgins, Annu. Rev. Cell. Biol. 8:67-113 (1992); Wijnholds, Novartis Found. Symp. 243:69-79, discussion 80-2, 180-5 (2002). Within these two families, two protein isoforms, one from each family, play a critical role in drug resistance. They are P-glycoprotein (Pgp or MDk1), which belongs to the ABC-B family (Gottesman and Pastan, Annu. Rev. Biochem. 62:385-427 (1993)), and MRP1, which belongs to the ABC-C family (Cole et al., Cancer Res. 54(22): 5902-10 (1994)). Pgp is the most extensively studied ATP-binding cassette transporter, functioning as a biological barrier by extruding toxic substances and xenobiotics out of the cells. In the clinical setting, the over-expression of multidrug resistance proteins such as MDR1 or MRP1 is often associated with poor prognosis for cancer patients.
Progress has been made in understanding of the structure-function relationship of some of the major drug transporters in recent years, notably the ABC transporters, and ABCB1 or MDR1 in particular. While certain amino acid residues in the drug and co-substrate (nucleotide) binding domains and those involved in the anchoring of the membrane-spanning polypeptides have been identified through site-directed mutagenesis and single nucleotide polymorphism studies, the actual biophysical mechanism of solute translocation is far from being elucidated.
The human MDR1 gene encodes a 170 kilodalton integral membrane protein that mediates ATP-dependent substrate efflux. The protein product, P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, resides in the plasma membrane and functions as an efflux transporter of a wide variety of natural compounds and lipophilic xenobiotics. While the contribution of P-glycoprotein in multidrug resistance for cancer chemotherapy is well documented, the role of P-glycoprotein in drug disposition is not fully understood and has continued to generate significant debate. P-glycoprotein mediates the energy-dependent efflux of a broad range of xenobiotics in epithelial tissues throughout the human body including the intestinal mucosa, liver canalicular membrane, kidney proximal tubules, blood-brain-barrier, and placenta. (Schinkel, Semin. Cancer Biol. 8:161-70 (1997)). Because P-glycoprotein is found in tissues important in drug disposition, variation in expression and function of P-glycoprotein due to genetic polymorphisms of MDR1 may influence pharmacokinetics and, in turn, pharmacodynamics.
Despite some similarities in drug resistance profiles, the MDR1 and MRP1 transporters differ somewhat in substrate selectivity, molecular structure, tissue distribution, and membrane location in cells. At the genetic level, MDR1 and MRP1 are only 15% identical in their amino acid sequences (Kruh et al., J. Bioenerg. Biomembr. 33(6): 493-501 (2001)). MRP1 is capable of transporting many lipophilic anions and conjugated substances. The MRP1 substrate includes a variety of structurally diverse anticancer drugs, GSH-conjugates, glucuronides, leukotriene C4 (LTC4), unmodified drugs, and some drugs that are multivalent organic anions, while Pgp substrates are mostly natural or mildly cationic molecules. A few previous studies (Paul et al., Biochem. 35(42): 13647-55 (1996); Stride et al., Mol. Pharmacol. 52(3): 344-53 (1997); Zh al. J. Biol. Chem. 276(16): 13231-9 (2001)) suggest that there are differences in substrate recognition and transport activity between the mouse and human ortholog of MRP1, indicating that significant interspecies differences exist for MRP1.
In rat, only a few reports are available for MRPs. It has been shown that the rat MRP1 expression occurs in almost all tissues, and its over-expression could confer multidrug resistance in cancer cells (Cherrington et al., J. Pharmacol. Exp. Ther. 300(1): 97-104 (2002)). Expression of rat MRP1 in the BBB capillary system and placenta has also been reported (Regina et al., J. Neurochem. 71(2):705-15 (1998)), suggesting that MRP1 plays certain roles in drug distribution into the central nervous system and in drug exchanges between the mother and fetus.
There are significant challenges in elucidating the functioning of transporters at the cellular and organ levels. Several reviews (Kunta and Sinko, Curr. Drug Metab. 5:109-124 (2004); van Montfoort et al., Curr. Drug Metab. 4:185-211 (2003)) have emphasized the concerted action of apical and basolateral membrane transporters in the translocation of a solute across epithelial barriers. The directionality of transport often requires carefully controlled studies with a cell barrier model. While cell barrier models for the intestinal and renal tubular epithelia are well-validated (e.g., Caco-2 LLC-PK1 and MDCK) and generally adopted, such models are still not available for the blood-brain barrier, the placenta, and the nasal and bronchial epithelia for direct evaluation of individual transporter proteins of interest such as MDR1 product Pgp and its genetic variants.
Research advances on the pharmacogenetics of human drug-metabolizing enzymes over the past two decades have afforded valuable insights into the clinical pharmacokinetics of a wide range of drugs, which have in turn led to clearer understanding of the metabolic basis of drug interactions and individual susceptibility to drug toxicities. It has had a profound impact on drug development and regulation.
The same impact may be observed as progress is made toward better understanding of the functional impact of genetic variations with polymorphically-expressed drug transporters. The genetic complexity of transporter genes is just beginning to be understood (see, e.g., Woodahl and Ho, Curr. Drug Metab. 5:11-19 (2004)). Linking the genetic polymorphism to a clinically discemable phenotype is complicated by linkage disequilibrium among single nucleotide polymorphisms. Improvement in molecular genetic technologies that could validate computational approaches that are currently used to assign and predict haplotypes could make a significant contribution in this area. Beyond the constitutive gene expression are issues related to gene regulation, such as induction and suppression of transporter gene transcription by endo- and xenobiotics, tissue-specific regulation through alternate splicing and post-translational modification, and modulation by diseases or pathophysiology.
Improved knowledge of drug transporters will optimize the delivery of a drug molecule to the target site(s) of interest. Discovery of new genetic variants of MDR1 that may have significant impact on drug penetration to cell and tissues as well as the availability of validated in vitro tools to predict drug availability and potential interactions with drugs that are given concomitantly could provide a significant improvement accelerating drug development and improve drug safety.